Under auspices of this R01 grant we have cloned the bHLH transcription factors Olig1 and Olig2. We have shown that these proteins regulate diverse functions in central nervous system development including specification of motor neurons and oligodendrocytes. The research described here builds upon progress made in the initial funding period. We have three specific aims: Specific Aim 1 is to define cis- and trans-acting regulatory elements that govern Olig2 expression in the embryonic spinal cord. Our initial focus is on elements driving Olig2 expression during neurogenesis. We have isolated a human Chr. 21-derived BAG clone that encompasses OLIG2 and approximately 116 kb of flanking material. Regulatory elements within this clone are sufficient to rescue motor neuron and oligodendrocyte specification in transgenic mice that are Olig2 null. Preliminary analysis leads to the testable hypothesis that TALE-HoxA class homeodomain proteins bind the enhancer resulting in Olig1 activation specifically in neuroblasts. Specific Aim 2 is to test the hypothesis that diverse neurogenic and gliogenic functions of Olig2 are regulated by phosphorylation. In preliminary studies we provide compelling evidence that a pair of protein kinase C (PKC) phosphorylation motifs in the carboxyl terminal domain control neurogenic functions of Olig2. We will knock phosphorylation state mutations of these PKC motifs into the wild type Olig2 locus and assess for biological function in developing mice. We will screen for additional phosphorylation sites that are biologically relevant using in ovo electroporation and state-of-art mass spectroscopy techniques. Specific Aim 3 is to define the role of Olig expression in glioma. In preliminary studies of more than 200 human brain tumors, we have noted that the Olig genes are expressed in 100 percent of human gliomas; moreover, it is the cycling cells within these tumors that are selectively Olig-positive. We will utilize contemporary genetic models of glioma to discriminate between three testable hypothesis: i) all gliomas arise from Olig-positive neural progenitor cells, ii) Olig genes are stringently required for proliferation/survival of glioma stem cells, iii) Olig1 and Olig2 are reporter genes for a signaling pathway common to all gliomas. The proposed work will shed light on fundamental mechanisms that regulate development of the normal brain from multipotent neural progenitor cells. Identification of regulatory factors for glial progenitor cell specification and differentiation will have practical overtones for the therapy of glial-based diseases such as multiple sclerosis and malignant glioma.